The role of genetic variants in the prediction of hearing loss due to cisplatin chemoradiotherapy

Abstract Background Concomitant high‐dose cisplatin with radiotherapy is commonly used for treating head and neck squamous cell carcinoma (HNSCC). Cisplatin, often used with radiotherapy, is known for causing irreversible sensorineural hearing loss, with individual variability suggesting a genetic component. This study aims to enhance the predictive ability of the clinical prediction model for cisplatin‐induced hearing loss (CIHL) in HNSCC patients, as outlined in Theunissen et al., by incorporating significant genetic variants. Methods Conducted at the Netherlands Cancer Institute, this retrospective study included 74 patients treated between 1997 and 2011. Thirty‐one SNPs that were previously associated with CIHL or other cisplatin‐induced toxicities were identified and incorporated into the model. The primary outcome measured was the change in decibels at posttreatment 1‐2‐4 kHz hearing levels per additional minor allele of these SNPs, evaluated using linear mixed‐effects regression models. The model's predictive accuracy was determined by the area under the curve (AUC) using 10‐fold cross‐validation. Results The rs2289669 SNP in the SLC47A1/MATE1 gene was linked to a significant 2.67 dB increase in hearing loss per allele (95% CI 0.49–4.86, p = 0.017). Incorporating rs2289669 improved the model's AUC from 0.78 to 0.83, a borderline significant improvement (p = 0.073). Conclusions This study underscores the importance of the rs2289669 SNP in CIHL and demonstrates the potential of combining genetic and clinical data for enhanced predictive models in personalized treatment strategies.


| INTRODUCTION
3][4] There is no standardized protective or curative agent available for neuro-and ototoxicity. 26][7][8][9] Recently, trans-tympanic sodium thiosulfate has also been studied as an otoprotector in a phase I study at the Netherlands Cancer Institute (NKI) and in a meta-analysis. 5,10isplatin has been known to instigate cochlear dysfunction, leading to sensorineural hearing loss (SNHL).2][13] CIHL is characterized by symmetric and irreversible SNHL starting at high frequencies, but after continued cisplatin courses it may progress to lower frequencies involved in speech perception. 2,14,15Other risk factors for the development of CIHL include favorable pretreatment hearing capacity, the use of other ototoxic drugs, and radiation exposure of the cochlea. 8,9,12,16,17lso, genetic variants have been associated with CIHL, such as single-nucleotide polymorphisms (SNPs) including ACYP2, [18][19][20][21][22] WFS1, 18,22 ABCC3, 23,24 and MATE1. 25heunissen and colleagues developed a prediction model for posttreatment hearing loss in patients with HNSCC who are treated with combined chemoradiation, see Figure S1. 26his model uses clinical input variables, such as baseline hearing thresholds, cisplatin dose, and cochlear radiation dose, to predict posttreatment hearing thresholds averaged at frequencies 1, 2, and 4 kHz.The model shows a 97% specificity and 29% sensitivity for the indication of hearing aids in the Netherlands (35 dB threshold). 26Despite the model being a significant step toward improving individual recommendations for HNSCC patients at risk for CIHL, the authors called for future research concerning additional risk factors.In particular, they hypothesized the role of genetic variants in hearing loss severity, based on observations of substantial individual vulnerability for the prevalence and severity of CIHL. 26,27Identifying SNPs linked to CIHL is crucial for personalized care, enabling better pretreatment guidance and interventions against chemoradiation side effects, including the use of trans-tympanic sodium thiosulfate for prevention.
The main aim of this study is to describe the contribution of genetics to individualized susceptibility to CIHL in an extension cohort of Theunissen et al. 26 and investigate whether using SNPs that are significantly associated with CIHL increases the predictive ability of our previously designed clinical prediction model. 26

| Data sources and patient selection
A retrospective cohort study was performed at the NKI from patients treated with intravenous (IV) cisplatin (100 mg/m 2 , for three courses) or with intra-arterial (IA) cisplatin (150 mg/m 2 ; for four courses, within the "RADPLAT trial") 28 during 7 weeks of radiotherapy (total dose of 70 Gy in 35 fractions on tumor-bearing areas) for advanced-stage HNSCC.All patients were treated between January 1, 1997 and December 31, 2011.Patients were selected based on available pre-and posttreatment audiometry and formalin-fixed paraffin-embedded tissue blocks.The patients treated with IA cisplatin have also been treated with concurrent IV sodium thiosulphate in order to prevent from platinum-related toxicity.We chose to also include these patients in the current cohort, as in these IA treated patients sodium thiosulphate did not protect against CIHL. 28The cochlear radiotherapy dose was also measured (methods described by Zuur et al. 17 ).All patients gave informed consent for further use of their data.

| Audiometry
Pure-tone audiometry was conducted pretreatment, 15-20 days after the first cisplatin infusion, and 3-31 weeks posttreatment in a sound-proof booth using the Decos Audiology Workstation.Audiometry consisted of pure tone audiometry (in hearing level (HL)) from 0.125 to 8 kHz, including both air conduction (AC) and bone conduction (BC), and ultrahigh frequency audiometry (in sound pressure level (SPL)) from 8 to 20 kHz.BC thresholds were used for the frequencies 0.5, 1, 2, and 4 kHz to correct for potentially fluctuating conductive components.Three pure tone averages (PTAs) were calculated: for the relatively low-frequency range relevant to quiet speech perception, we calculated PTA 0.5-1-2 kHz HL BC (PTAL).For the relatively high-frequency range relevant to speech perception in noise, we used PTA 1-2-4 kHz HL BC (PTAH).For the perception of ultrahigh sounds (e.g., music or nature), we investigated PTA 8-10-12.5 kHz SPL AC (PTAU).Missing audiometric data at the PTAs at baseline and after the first cisplatin cycle were imputed following the method described previously by Theunissen et al., 26 while patients with missing posttreatment audiometry were excluded.

| Isolation of DNA and sequencing
DNA was isolated from FFPE tissue blocks at NKI. Next, the Princess Margaret Cancer Centre in Toronto performed sequencing of 31 SNPs by MassArray SNP genotype method (Sequenom) using Assay Design Suite software.

| Gene selection
The genetic variants were selected based on published literature, had a minor allele frequency in individuals of European descent of at least 1%, and had the ability to incorporate the polymorphism or a surrogate in high (D' >0.95) linkage disequilibrium in the multiplex reaction.Included SNPs and associated references from literature review can be found in Table S1.
In our analysis, certain SNPs identified as significant in previous literature were not available in our genotyping panel; therefore, we selected proxy variants based on linkage disequilibrium data and prior association studies.Specifically, rs1051740 was used as a proxy for rs1142345, rs2273697 for rs1800462, and rs4646316 for rs11568591.

| Statistical analysis
Descriptive statistics were used to summarize baseline patient characteristics and allele and genotype frequencies of the 31 SNPs of interest.
Multivariate imputation was performed using the pan-Impute function from the mitml package in R to account for missing data.Missing audiometry data at baseline (4 ears [3.5%] for PTAL and 4 ears [3.5%] for PTAH), and after the first cisplatin infusion (57 ears [49.6%] for PTAL, 57 ears [49.6%] for PTAH, and 31 ears [27.0%] for PTAU), were imputed.Imputations were functions of the outcome (i.e., posttreatment PTAs) as well as PTAs at baseline and first chemotherapy, chemotherapy dosage and cochlear radiation dose.PTAs at baseline, the first cisplatin infusion, and posttreatment were log-transformed to ensure normally distributed residuals where necessary and backtransformed for use in the main model of interest.
Quality control analysis was performed on the genetic data.This included the exclusion of SNPs without variation in allele frequency.The rs77382849 SNP (EIF3A) was excluded from the analysis, as all patients were observed to have the homozygous major genotype, rendering it uninformative for association analysis.Additionally, the Hardy-Weinberg equilibrium was assessed, and no SNPs violated the assumption (p-value>0.05).
CIHL may differ in both ears, as the ear ipsilateral to the tumor may receive a higher dose of radiation compared with the contralateral ear.Thus, the outcome was expressed per ear on two occasions: left and right ears after the first cisplatin infusion and the left and right ears after the end of treatment.To determine if any of the candidate SNPs were independently associated with posttreatment hearing capability, linear mixed-effects models on posttreatment PTA with patient-specific intercepts were run for one SNP at a time.
We created two sets of models: 'Unadjusted models' evaluated the association of a SNP with posttreatment hearing capability adjusted for pretreatment highfrequency PTA (PTAH) only.'Adjusted models' evaluated the association of a SNP on the same posttreatment hearing capability adjusted for all pretreatment (low, high, and ultra-high) PTAs (PTAL, PTAH, PTAU), cumulative chemotherapy and radiation dose to the cochlear.We adjusted for the same clinical factors as those used in the original model by Theunissen et al.To account for additive gene effects, the genotype with the major allele homozygous was coded as '0', the heterozygous genotype as '1', and the genotype with the minor allele homozygous as '2'.Pooling of results of 100 imputed datasets was performed using the summary function (testEstimates) from the mitml package.Unadjusted and adjusted p-values were computed without correction for multiple testing; rather than dismissing associated SNPs to control family-wise error rate, we included significant SNPs with a liberal p-value threshold to preserve any potentially clinically important SNPs in the final model.
To ensure consistency in findings, a sensitivity analysis was performed using co-dominant, adjusted linear mixed-effects regression models.For this model, patients with two major alleles in a given gene were coded as zero, heterozygous patients were coded as 1, and patients with two minor alleles were coded as 2. We utilized the codominant genetic model due to its flexibility and detailed categorization of genotypes.
We performed a likelihood ratio test (LRT) to determine the goodness of fit of the two models, with and without the SNP.Model comparisons were calculated and summarized over the 100 imputed datasets.
To evaluate and compare the performance of the new prediction model to predict observed PTAH of at least 35 dB de novo (the Dutch threshold for hearing aid qualification) to the original prediction model, we computed the 10-fold cross-validated sensitivity and specificity of both models at all possible thresholds of predicted HLs.Cross-validation was performed at the patient level.
Sensitivity and specificity for each cut-off were imposed on the model predictions to classify patients as "hearing loss" (predicted hearing at/above cut-off in either ear) versus "no hearing loss" (predicted hearing below cut-off in both ears).Patients with missing data for any identified statistically significant SNP associated with the outcome, or those who had baseline PTAH ≥35 dB (therefore classified as "hearing loss" before treatment) were removed from cross-validation.We then constructed the receiver operating characteristics (ROC) curve by plotting 10-fold cross-validated sensitivity versus 1-specificity and calculated the area under the curve (AUC) with 95% confidence intervals.To assess the discriminatory performance of the models, we performed DeLong's test to compare the ROC curves of the new and the original prediction model to statistically evaluate and compare the predictive accuracy of the two models.
Data were analyzed using R software (v.2022.12.0).All tests were two-sided and α = 0.05 was used to denote statistical significance.

| Patient selection
A total of 115 HNSCC patients received high-dose chemoradiation as a primary treatment and had genetic SNP and audiometry data available.If a patient had complete data for only one ear, that ear was retained for analysis.We excluded 35 ears with missing radiation dose to the cochlea (both ears from 17 patients, one ear from 1 patient), and 54 ears without posttreatment PTAH (both ears from 24 patients, one ear from 6 patients).In total, 74 patients (64.3%) and 141 ears were included (Figure S2).Due to the unavailability of tissue samples for some subjects from Theunissen et al.'s cohort, our current cohort differs slightly.Additionally, it includes 18 patients who underwent treatment with IA cisplatin.

| Descriptive analysis
Table 1 summarizes the patient sample.The mean age of participants was 53.9 (SD = 8.5) years and 77% (n = 57) of participants were male.The cumulative cisplatin dose among patients ranged from 315 to 1200 (median = 579.5)mg.The radiation dose to the cochlea ranged from 1.1 to 70.5 Gy (median = 11.4Gy) because the patients were, in general, treated with intensitymodulated radiotherapy.
For the majority of patients, as time progressed from the first cisplatin infusion to posttreatment, PTAH (kHz) increased over time (i.e., hearing loss occurred) (Figure 1).T A B L E 1 Descriptive statistics of patient population (n = 74) demographics, chemotherapy, and radiation dose to the cochlea.

| Gene analysis
Of the 30 candidate SNPs, only one was a statistically significant predictor of posttreatment hearing capability.The rs2289669 SNP (within gene SLC47A1/MATE1) showed a 2.67 dB (95% CI 0.49-4.86,p = 0.017) greater hearing loss for each additional minor allele (Table 2).

| Sensitivity analysis
In a sensitivity analysis, regression models were reanalyzed using a co-dominant model.The rs2289669 SNP (particularly the homozygous minor AA genotype) was again the only significant predictor of posttreatment hearing capability, consistent with our additive gene model findings (Table S3).

| Model evaluation
The inclusion of the rs2289669 SNP to our preexisting clinical prediction model including baseline PTA, chemotherapy dose and radiation dose, significantly improved its goodness-of-fit (LRT p = 0.017).Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) at different hearing cut-offs for both models ("SNP model" with the SNP and "clinical-only model" with clinical factors only) are displayed in Table 3.The addition of rs2289669 improved sensitivity and specificity at a threshold of 30 and 35 dB (sensitivity 0.44 vs. 0.38; specificity 0.96 vs. 0.94) but not at 25 and 40 dB.We performed a supplementary analysis to observe changes in predictive ability with the addition of the top 10 SNPs, chosen based on significance (i.e., p-values).We found that adding 10 more SNPs to the model did not improve the predictive ability, and, due to substantial overfitting, resulted in a decrease in the AUC.
The SNP model had a moderately, borderline significant (p = 0.073), higher AUC (AUC = 0.83, 95% CI 0.71-0.94)compared to the clinical-only model (AUC = 0.78, 95% CI 0.66-0.91).The observed predictive gains were not consistent across all hearing thresholds, with the SNP model demonstrating the greatest advantage at the 30 dB HL, as evident from the ROC curve plots (Figure 2).

| DISCUSSION
This study aimed to enhance the predictive ability of the clinical prediction model for CIHL designed by Theunissen et al. by adding genetic information.We F I G U R E 1 Spaghetti plot of pure tone audiometry at high frequencies (PTAH) in kHz, a measure of hearing loss, over time, from baseline to posttreatment.C1: first cisplatin dose.Time between baseline and C1 was 3 weeks, time between C1 and posttreatment was median 14 weeks.H+-coupled organic cation bidirectional antiporter, expressed on the apical membrane of the tubular epithelium of the kidneys. 25,32The human tissue distribution of MATE1 is comparable to that in mice, where it has been shown to play an important role in the pharmacokinetics of several drugs. 25Nakamura et al. found that the plasma and renal levels of cisplatin were significantly higher in MATE1 knock-out mice than in wild-type mice. 31MATE1 likely interacts with the organic cation transporter 2 (OCT2) in the systemic distribution of cisplatin. 29,32OCT2 was already reported to be expressed in the cochlea and to be involved in the cochlear uptake of platinum.Interestingly, very recently, Waissbluth et al.
showed that MATE1 is expressed in the inner ear, and that cisplatin decreases both OCT2 and MATE1 expression in the cochlea. 30hat is known about the MATE1 rs2289669 variant from a study with patients using metformin-of which the uptake is also regulated by MATE1-is that it is likely associated with reduced transporter function. 33A mice study demonstrated that MATE1 deficiency leads to increased cisplatin-related nephrotoxicity and hematological toxicity (OR = 1.92, 95% CI 1.13 to 3.25, p = 0.016). 32Hence, if MATE1 rs2289669 is indeed associated with reduced transporter function, this variant may also be associated with a higher risk of CIHL.This is supported by our results, as we found that the A-allele is associated with more hearing loss.However, this contradicts Teft et al.'s findings that the rs2289669 MATE1 A/A homozygous variant significantly reduces CIHL risk in 206 HNSCC patients (HR = 0.46, 95% CI 0.26-0.84). 25The exact role of MATE1 in the cochlea needs to be further studied, although this may be challenging due to its bidirectional properties. 25r analysis did not show significant associations between CIHL and several SNPs (TPMT, 23 ABCC3, 23,24 COMT, 24,25,34 WFS1, 18,22 ACYP2, [18][19][20][21][22][34][35][36] ERCC2, 34 XPC, 34 and GSTP1 34 ) as previously reported. Th0 Additionally, many studies did not assess ultrahigh frequencies (8.0-20.0 kH SPL), which generally are more sensitive frequencies for identifying CIHL, considering its initial manifestation at these frequencies.
This study benefits from a well-defined cohort of patients who received combined chemoradiation as a primary treatment for HNSCC.The inclusion of SNPs and audiometry data allowed for a comprehensive analysis of the relationship between genetic variants and CIHL.Our study adopted a targeted approach by including 30 candidate SNPs in the model-building process, strategically focusing on specific genetic markers of interest, based on published literature.
A limitation of the current study is that it relied on a retrospective analysis.Information on chemo-and radiotherapy dosage, audiometry, and other clinical factors was missing in a small number of patients.To address potential data gaps, we employed a mechanism of multiple imputation based on chained equations.Second, the selection of the 31 SNPs in this study was informed by a review of literature available up until 2017.Therefore, any genetic markers linked to ototoxicity discovered post 2017 were not included in our analysis.This could mean that potentially relevant SNPs identified after this cutoff were not included in the analysis.Finally, the predictive ability of the model, despite the inclusion of genetic variants, still demonstrated limitations in sensitivity and specificity.This suggests that factors beyond the current set of variables, such as environmental factors or additional genetic markers, may contribute to the variability in CIHL susceptibility.
CIHL is a common and debilitating side effect of cisplatin treatment, with important consequences on patients' quality of life. 8Understanding the genetic factors contributing to CIHL may enhance clinical prediction models, allowing clinicians to provide tailored pretreatment counseling, inform patients about the potential risk of CIHL, and enable the identification of individuals who may benefit from otoprotectants.Moreover, the identification of SNPs related to CIHL may facilitate the development of novel preventive strategies and interventions.Additional research is needed to assess the validity and generalizability of the prediction model in various genetic backgrounds and clinical settings in other HNSCC patient populations.Furthermore, the specific mechanisms through which these genetic variants contribute to the development of CIHL need to be elucidated.

| CONCLUSION
Our findings demonstrate a significant association between the rs2289669 SNP within the SLC47A1 (MATE1) gene and posttreatment hearing capability in patients undergoing combined chemoradiation.These results contribute to a deeper understanding of the genetic factors influencing hearing outcomes in this population and provide insights into potential strategies for personalized treatment approaches in the future.

F I G U R E 2
Receiver operating characteristics (ROC) curve for models with (SNP model) and without (clinical-only model) incorporation of the rs2289669 SNP.
[29][30][31][32]djusted additive, linear mixed-effects regression model results for each of the 30 SNPs of interest.Recently, the role of MATE1 in the development of cisplatin toxicity was studied.[29][30][31][32]MATE1 is an Model coefficients are interpreted as the change in follow-up PTAH for each additional minor allele.For example, each additional minor allele of the rs2289669 SNP was associated with a 2.67 dB increase in follow-up PTAH Lower values of PTAH indicate better hearing capability.hearing loss from these factors plus rs2289669 genotype.Bolded rows are where the SNP model has improved performance over the clinical-only model.
gests that individuals with less frequent variants of this SNP (i.e., homozygous minor) are more likely to develop CIHL, compared to those with more common variants.For context, in our cohort, 17.8% of individuals carried the homozygous minor allele for rs2289669, highlighting the potential impact of these rarer variants.When *Significant or borderline significant SNPs.a b Unadjusted models include adjustment for pretreatment PTAH only.c Adjusted models include adjustment for all pretreatment (low, high, and ultra-high) PTAs (PTAL, PTAH, PTAU), chemotherapy dosage, and radiation dose to the cochlea.T A B L E 2 (Continued) T A B L E 3 Sensitivity and specificity at different hearing cut-offs (25-40 dB). a Clinical-only model predicts hearing loss from baseline PTA, chemotherapy dose, and cochlear radiation dose.b SNP model predicts